ABSTRACT A theme of our research program is the design and assembly of symmetric surrogates of the quasi- hexagonal and pleiomorphic lattices of the HIV-1 immature and mature capsids that enable high-resolution structure analysis by cryoEM, microED, X-ray crystallography and ssNMR spectroscopy. Interpretation of our experimental results is fortified by the addition of multi-scale, computational simulations. Aim 1: Structural studies of the immature Gag lattice: We previously determined a 3.2 X-ray structure of a CTD-SP1 Gag construct, which revealed a 6-helix bundle comprised of 2 turns of CTD and 2 turns of SP1. The protease cleavage sites are sequestered in the interior of the bundle. This surprising result revealed a mystery: How does protease gain access to the sequestered cleavage sites? ssNMR spectroscopy of selectively labeled ?MA-Gag VLPs will allow us to examine conformational dynamics of the junction helices. Time-resolved cryoEM of 2D crystals of CTD-SP1, which diffract to 5- resolution, will test whether protease cleavage is initiated at the fissures within the immature Gag lattice. MicroED of 2-4 m 3D crystals yielded a 2.9- resolution 3D structure of CTD-SP1 with bound bevirimat, which sets the stage for us to explore mechanisms of drug resistance and action of second-generation maturation inhibitors. Our results will not only provide data for atomic-resolution MD simulations, but also help develop and refine novel coarse-grained (CG) models of Gag lattice components to explore the dynamics of on- and off-pathway generation of viral particles. Aim 2: Structural studies of the mature capsid: We previously used disulfide crosslinking to stabilize and solve X-ray structures of the CA hexamer and pentamer. The resulting atomic model of the fullerene cone suggested mechanisms for the continuously varying curvature in the conical capsid. We also showed that the compound PF74, a CPSF6 peptide and a peptide of NUP153 bind the interface between the C-terminal and N- terminal domains of CA, indicating that this interface is a therapeutic target and may be important for docking of the capsid at the nuclear pore. We will now explore a surrogate of capsid docking at the nuclear pore by examination of 2D CA crystals with bound NUP153, allowing us to test whether the FG repeats bind cooperatively and whether binding disrupts the lattice to release the preintegration complex into the nucleus. We have shown that the R18F mutant of CA forms ~35 nm spherical particles, and a preliminary 3D cryoEM reconstruction shows that the ~90 spacing between protomers in the icosahedral lattice recapitulates the spacing in authentic HIV-1 capsids. We seek to determine an atomic resolution structure to examine the chemical interactions at the protomer-protomer interfaces. The 7- cryoEM map of mature HIV-1 and the high- resolution map of the icosahedral surrogate will enable computational simulations of the mature capsid lattice to investigate the effects of protein and drug binding on lattice self-assembly and stability. We believe our studies will yield novel insight into HIV-1 maturation and assembly that will be relevant for drug discovery.